Staphylococcus aureus colonizes the skin and mucosal membranes of humans and animals, and together with the other members of the genus Staphylococcus, has been implicated in a diverse array of infections. S. aureus contains many virulence factors including surface proteins designated as “microbial surface components recognizing adhesive matrix molecules,” which facilitate attachment to surfaces and initiate infection (Gordon & Lowy (2008) Clin. Infect. Dis. 46:S350-5359). S. aureus can also form biofilms (Donlan & Costerton (2002) Clin. Microbiol. Rev. 15:167-193), which allow it to evade both the immune system and antibiotics. Most strains have a polysaccharide capsule and secrete a variety of enzymes that are used during infection to enhance bacterial spreading (Foster (2005) Nat. Rev. Microbiol. 3:948-958). S. aureus can also cause toxic shock syndrome, and studies have shown that the peptidoglycan and lipoteichoic acid of the S. aureus cell wall act together to cause toxic shock in rats (Kimpe, et al. (1995) Proc. Natl. Acad. Sci. USA 92:10359-10363).
Antibiotic resistance in staphylococci appeared after penicillin was first used for treatment of staphylococcal infections. This development of resistance, which was present in over 80% of clinical isolates by the late 1960s (Lowy (2003) J. Clin. Invest. 111:1265-1273), prompted the development of new, more potent drugs to combat the opportunistic pathogen. These efforts led to production of methicillin, a narrow spectrum penicillinase-resistant drug designed to alleviate the burden of staphylococcal infections. However, it took only a year for the first methicillin-resistant S. aureus (MRSA) clinical isolates to be discovered.
Initially, MRSA infections were only associated with prolonged hospital treatment and invasive surgical procedures, and were classified as Health Care-Acquired MRSA (HCA-MRSA). However, in recent years, MRSA has also emerged as a community-acquired infection (CA-MRSA), which affects groups with high-intensity physical contact, such as competitive athletes, military recruits, and children in daycare centers (Romano, et al. (2006) J. Athl. Train. 41:141-145; Kazakova, et al. (2005) New Engl. J. Med. 352:468-475; Zinderman, et al. (2004) Emerg. Infect. Dis. 10:941-944; Adcock, et al. (1998) J. Infect. Dis. 178:577-580).
The S. aureus cell wall is composed of alternating polysaccharide subunits of N-acetylglucosamine and N-acetylmuramic acid, wherein each N-acetylmuramic acid is connected to a peptide chain. Cross-linking of the peptidoglycan is achieved by four major penicillin-binding proteins (PBP1, 2, 3 and 4) that connect the muropeptide chains via pentaglycine interpeptide bridge. Methicillin resistance arose when S. aureus acquired the mecA gene, which encodes for penicillin-binding protein PBP2A that has transpeptidase activity but lower affinity for penicillin and β-lactam antibiotics. Resistant cells still produce PBPs, but given the expression of PBP2A, peptidoglycan synthesis continues in the presence of methicillin and other β-lactams (Hiramatsu, et al. (2001) Trends Microbiol. 9:486-493).
Lysostaphin is a glycyl-glycine zinc-dependent endopeptidase produced by Staphylococcus simulans, which selectively targets pentaglycine interpeptide cross-bridges. The gene for lysostaphin has been isolated and characterized. Genetic truncations have been made to remove the 36-residue signal Peptide and 224-residue long propeptide thereby facilitating fusion to either an initiating methionine for intracellular expression or an exogenous signal sequence, e.g., to permit the secretion of a single species of lysostaphin into the periplasmic space of E. coli (See, e.g., US 2005/0118159). The mature, 247-residue enzyme is composed of N-terminal catalytic domain (138 amino acids), which is connected to the C-terminal cell wall binding domain (92 amino acids) via an 18-residue linker (Lu, et al. (2013) Antimicrob. Agents Chemother. 57:1872-1881).
Lysostaphin has shown promise as a therapeutic agent for treatment of S. aureus infections. The protein has been shown to lyse staphylococcal strains (Schindler & Schuhardt (1964) Proc. Natl. Acad. Sci. USA 51:414421) and clinical isolates (Cropp & Harrison (1964) Can. J. Microbiol. 10:823-828), and demonstrated remarkable efficacy in animal models (Schuhardt & Schindler (1964) J. Bacteriol. 88:815-816; Schaffner, et al. (1967) Yale J. Biol. Med. 39:230-244; Goldberg, et al. (1967) Antimicrob. Agents Chemother. 7:45-53; Kokai-Kun, et al. (2007) J. Antimicrob. Ther. 60:1051-1059; Placencia, et al. (2009) Ped. Res. 65:420-424; Climo, et al. (1998) Antimicrob. Agents Chemother. 42:1355-60), including those of staphylococcal biofilms (Kokai-Kun, et al. (2009) J. Antimicrob. Ther. 64:94-100). In several of these studies, antibodies against lysostaphin were observed in animals subjected to the drug for a prolonged period of time (Climo, et al. (1998) Antimicrob. Agents Chemother. 42:1355-1360). Similarly, human clinical trials with intranasal lysostaphin indicated a slight elevation in anti-lysostaphin antibody titer (Kokai-Kun (2012) in Antimicrobial Drug Discovery: Emerging Strategies (Tegos & Mylonakis, eds) Ch. 10, 147-165).
In an attempt to improve pharmacokinetics and reduce immunogenicity, lysostaphin has been linked to branched polyethylene glycol (PEG). While PEGylation reduced immunoreactivity, PEGylation of the enzyme significantly reduced its activity (Walsh, et al. (2003) Antimicrob. Agents Chemother. 47:554-558). In addition, US 2008/0095756 describes the deimmunization of the cell wall binding domain of lysostaphin. However, variants with a deimmunized catalytic domain are not described.